(Project 2) A marked decline in proteasome activity is observed as humans and other mammals age. This has been observed in many tissues, including the brain and motor neurons. Aging is characterized by compromised proteostasis, and declining proteasome activity may play a significant role in aging-associated deficiencies of proteostasis, given the pivotal role of the proteasome in protein dynamics. By degrading ubiquitin conjugates, the proteasome controls protein stability on a global level. There has historically been little interest in the potential role of the proteasome in determining the overall output of the ubiquitin-proteasome pathway (UPS). Through recent work, however, it is now recognized that the proteasome is on the contrary a focal point of regulation of the UPS. Indeed, the level of proteasome activity controls protein breakdown rates and stress resistance. Remarkably, the proteasome can in general be up-regulated without toxicity. Extensive work has also shown that the proteasome is compromised in many disease states, particularly in aging-associated and neurodegenerative diseases. However, a deeper and more reliable understanding of the relevance of the proteasome to aging and neurodegeneration in humans clearly requires the use of in vivo mammalian model systems. To directly test whether the proteasome becomes limiting in aged animals, three transgenic mouse lines have been designed to allow conditional elevation of proteasome levels; two of these lines have already been generated. Each mouse line will conditionally express the wild-type murine form of a different proteasome subunit?either Rpn6, Rpn11, or ?5. These subunits were chosen because they are expected from existing data to be rate-limiting for proteasome assembly. The multiplicity of strategies to elevate proteasome levels increases the likelihood of success, and if more than one method succeeds it will enhance confidence in subsequent results. For each transgene, a floxed and dox-suppressible construct is integrated into the Rosa26 locus via CRISPR/Cas9. The transgenes should be expressed in a tissue-specific and temporally-controlled manner. Our first objective will be to validate the strategy for elevating proteasome levels in the brain, spinal cord, and in Flp-In?-3T3 cells with transgenes similarly targeted to Rosa26. Excellent biochemical and proteomic methods exist for quantifying alterations in proteasome levels. Remarkably, global proteomics can now quantify the impact of elevated proteasome levels on the control of hundreds of substrate proteins in these settings. We will proceed to assess the effects of elevated proteasome expression on the health and aging of these animals. We will test whether elevated proteasome levels influence autophagy and the proteostasis network (PN) as a whole, and seek to identify age-dependent vulnerabilities in the PN by applying specific stresses to the system. A central focus of the work on transgenic mice will be to employ disease models to assess the effect of elevated proteasome levels on the progression of neurodegenerative diseases, particularly tauopathies, Huntington?s disease, and ALS.